The siRNA molecule needs better protection from nuclease degradation before it can be effectively used as a therapeutic. AM Biotechnologies (AM) will address this critical issue by developing ribonucleoside thiophosphoramidite (ABz, CBz, GIbu and U) reagents that will enable synthesis of phosphorodithioate siRNA (PS2-siRNA). This synthesized PS2-siRNA will significantly increase siRNA nuclease resistance;lower Tm, which could help the activated RISC unwind siRNA;and enhance the pharmacokinetic properties of siRNA. The PS2-siRNA will also be achiral at phosphorus, which will eliminate the variable biochemical, biophysical, and biological properties of diastereomeric phosphoromonothioate substituted siRNAs (PS-siRNAs). In this Phase I SBIR project, AM will: 1) develop the chemistry and optimize the conditions to produce four ribonucleoside thiophosphoramidites (ABz, CBz, GIbu and U);2) demonstrate high coupling yield (>97%) in the synthesis of PS2-siRNAs;and 3) evaluate PS2-siRNA gene silencing effect in vitro. In Phase II, AM will perform the research required to (a) scale reagent production up to commercial quantities and purity;(b) optimize a robust protocol for synthesis of PS2-siRNA;(c) systematically evaluate the positional effect of PS2 modification on siRNA activity in mammalian cells;(d) examine the bio-distribution of PS2-siRNA;and (e) fully characterize the pharmacokinetic properties of PS2-siRNA. AM in Phase II may also offer for sale limited quantities of research-grade reagents for market beta testing. Upon successful completion of Phase II, AM will work with its industry partners to commercialize the ribonucleoside thiophosphoramidites (R-thioamidites) and enable the entire life science community to benefit from the use of these unique reagents.